Engagement of multicomponent immunoreceptors such as the T cell antigen receptor (TCR) results in rapid activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. Our studies have focused on two substrates of these PTKs, LAT (linker for activation of T cells) and Cbl. LAT is a 36-38kD integral membrane protein. We have been able to purify this molecule, and have cloned the cDNA that encodes it. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Two independently derived mutants of the Jurkat human T cell line have been shown to be deficient in LAT. TCR crosslinking in these cells fails to result in any indication of T cell activation. However, reconstitution of LAT completely restores TCR-mediated activation in these cells, thus demonstrating the importance of this molecule for TCR signaling. LAT-deficient mice have also been generated using standard gene targeting procedures. T cell development in these mice is blocked at an early stage within the thymus. Thus this complex developmental pathway is also dependent on LAT. Three other cell types, mast cells, NK cells and platelets, express the LAT protein. Mast cells derived from the LAT-deficient mice develop normally. However engagement of the mast cell immunoreceptor, Fc"Symbol"eRI, fails to induce optimal degranulation or cytokine production. NK cells and platelets also demonstrate a dependence on LAT. The cytotoxic activity of some NK clones can be blocked by dominant negative LAT expression, and platelet function mediated by the platelet GPVI receptor fails to occur in LAT-deficient platelets. Current studies on LAT function focus on the role of individual tyrosine residues. Tyrosine to phenylalanine mutations have been introduced, and these mutant molecules have been re-expressed into LAT-deficient cells and mice. One other critical PTK substrate in T cells is the Cbl molecule. Studies this year have demonstrated that it regulates T cell activity in a unique fashion. One of its structural features, an SH2 domain, binds to tyrosine phosphorylated PTKs, while another domain, a ring finger, appears to be involved in PTK down-regulation. Forms of Cbl that contain mutations in the ring finger, when expressed in T cells are activating. Our studies indicate that these mutations thus have a dominant negative effect on PTK regulation.(Project Number Z01-HD-01600-14 CBMB was transferred from CBMB, NICHD to LCMB, NCI)